Project Summary The malaria parasite is one of the most deadly eukaryotic pathogens, causing over 400,000 deaths in 2014, 78% of which occurred in children less than 5 years old. Due to growing resistance to currently available medications, there is a pressing medical need for new drugs to prevent and treat malaria infection. An ideal antimalarial drug would target biochemical pathways that are absent in human. Such a drug would be expected to have excellent safety characteristics, especially for children and pregnant women, who represent the most susceptible populations. We have identified a compound 1a (MMV008138) that inhibits Plasmodium falciparum IspD (PfIspD), the third enzyme in the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid precursor biosynthesis, and inhibits growth of the parasite in vitro (IC50 = 250 70 nM). Since isoprenoid precursors are essential for parasite survival, and since humans synthesize them by the mevalonate pathway, compounds like 1a could provide the basis for a new safe and effective therapeutic strategy for malaria. In the R21 phase of this proposal, we will obtain high resolution X-ray crystal structures of PfIspD, to locate the binding site of 1a on the enzyme, and to understand the structural determinants of PfIspD inhibition potency. In parallel, we will use two methods to search for new inhibitors of PfIspD. First, we will leverage our collection of P. falciparum growth inhibition data for 92 close analogs of 1a, to carry out atomic property field-based virtual ligand screening (VLS) of a library of 5 million publicly available compounds. Secondly, we also prepare new bisubstrate analog inhibitors of PfIspD by linking mimics of the two substrates of PfIspD, MEP and CTP. Compounds identified by VLS and bisubstrate analog inhibitors will be screened for inhibition of PfIspD and P. falciparum growth. In the R33 phase of the proposal, the X-ray crystal structures of PfIspD obtained will be used to optimize the antimalarial efficacy of 1a and the new compounds identified in the R21 phase. Compounds meeting minimum potency and selectivity thresholds will advance to ADME-Tox and pharmacokinetic studies (mouse). The best compounds emerging from these studies will be evaluated for in vivo efficacy in a mouse model of malaria. Efficacious compounds identified in this way will thus be well-positioned for further preclinical development.